The invention relates to the field of biotechnology. More in particular the invention relates to the field of antibodies and uses thereof. One of such uses relates to medical uses of antibodies.
The exposure to a highly diverse and continuously changing environment requires a dynamic immune system that is able to rapidly adapt in order to adequately respond to potentially harmful micro-organisms. Higher organisms have evolved specialized molecular mechanisms to ensure the implementation of clonally-distributed, highly diverse repertoires of antigen-receptor molecules expressed by cells of the immune system: immunoglobulin (Ig) molecules on B lymphocytes and T cell receptors on T lymphocytes. For B lymphocytes, a primary repertoire of (generally low affinity) Ig receptors is established during B cell differentiation in the bone marrow as a result of rearrangement of germline-encoded gene segments. Further refinement of Ig receptor specificity and affinity takes place in peripheral lymphoid organs where antigen-stimulated B lymphocytes activate a somatic hypermutation machinery that specifically targets the immunoglobulin variable (V) regions. During this process, B cell clones with mutant Ig receptors of higher affinity for the inciting antigen are stimulated into clonal proliferation and maturation into antibody-secreting plasma cells (reviewed in 1).
In recent years, recombinant DNA technology has been used to mimic many aspects of the processes that govern the generation and selection of natural human antibody repertoires (reviewed in 2,3). The construction of large repertoires of antibody fragments expressed on the surface of filamentous phage particles and the selection of phages by panning on antigens has been developed as a versatile and rapid method to obtain antibodies of desired specificities (reviewed in 4,5). Further optimization of the affinity of individual phage antibodies has been achieved by creating mutant antibody repertoires that are expressed on bacteriophage particles and sampled for higher affinity mutants by selection for binding to antigen under stringent conditions (reviewed in 6). Various approaches have been used to create mutated antibody repertoires, including chain shuffling (7, 8), error prone PCR (9), use of E. coli mutator strains (10) or approaches more specifically directed to the complementarity determining regions (CDRs) of the antibody molecule, like CDR walking and parsimonious mutagenesis (11-13).
To select higher affinity mutants from a library of phage-displayed, mutagenized antibody fragments, selections have been performed on purified immobilized antigen or biotinylated antigen in solution, followed by capture of phage bound on streptavidin magnetic beads (14-16). It has been demonstrated that the selection of higher affinity single chain Fv antibody fragments (scFv) specific for the antigen c-erb-2 from phage libraries of mutants of that scFv was dependent on the availability of purified antigen in solution. Antigen captured on a solid phase resulted in the isolation of false positives with higher avidity rather than affinity due to the dimerization and oligomerization of the scFv on the phage. In addition, it was shown to be crucial for the isolation of higher affinity scFv to perform subsequent rounds of phage selections with carefully controlled and increasingly lower antigen concentrations in solution (14). Although very high-affinity scFv have been isolated with these approaches, they are not readily applicable when the target antigen is difficult to express as a recombinant molecule or tedious to purify in sufficient quantities without losing its native configuration. Examples of these types of molecules are seven-transmembrane spanning proteins, insoluble lipid-modified membrane molecules and post-translationally-modified proteinaceous molecules that are specific for particular cell types or disease states. Thus a selection procedure for higher affinity mutant antibody fragments, without the need for purified antigen would represent an important extension of affinity maturation strategies for phage displayed antibodies.
The invention now in one aspect provides a method for selecting a member from a library of proteinaceous molecules comprising providing cells and/or a functional equivalent thereof, with at least part of said library under conditions that allow binding of any member to an epitope in and/or on said cells and/or said functional equivalent thereof, removing unbound proteinaceous molecules and selecting said member, wherein said library comprises at least one mutant of a proteinaceous molecule capable of binding to said epitope. Preferably a mutant comprises one or more mutations that affect the capability of binding of the mutant to said epitope in a positive or negative way, compared with the unmutated proteinaceous molecule. The capability may be affected by an altered binding affinity or altered dissociation constant, or both.
A member of the library is a proteinaceous molecule present in said library and/or a proteinaceous molecule selected from said library. A selected member typically comprises the capacity to bind to said epitope. Once selected and characterized a member may also be generated in another way for instance artificially, through molecular biological techniques such as but not limited to peptide synthesis or the expression of a nucleic acid encoding said proteinaceous molecule. A proteinaceous molecule may be a peptide, a polypeptide or a protein. Peptides are strings of amino acids linked together by a peptide bond. Although not precisely defined, peptides typically comprise between 2 and 50 amino acids. Polypeptides are longer peptides that may contain up to thousands of peptide bond-linked amino acids. The words polypeptide and protein are often interchangeably used to describe single, long polypeptide chains. In addition, proteins may consist of multiple polypeptide chains that collectively form the basis of a complex three-dimensional structure. A peptide, a polypeptide and/or a protein may comprise modifications such as those generated by a cellular protein modification machinery. A mutant of a proteinaceous molecule is a proteinaceous molecule comprising one or more changes compared to the unmutated proteinaceous molecule. A change can comprise for instance an exchange, a deletion, an insertion or an addition of one or more amino acids or a combination of these changes. Preferably but not necessarily said mutation is generated through a change in a nucleic acid encoding said proteinaceous molecule.
A library comprises at least one mutant of a proteinaceous molecule capable of binding to an epitope. Typically, a library will comprise more than 100 different mutants of said proteinaceous molecule. Such a library may be used on its own or it may be combined with one or more other libraries comprising at least one mutant of another proteinaceous molecule capable of binding to at least a part of said epitope. An advantage of such a combination is that it increases the complexity of mutants thereby increasing the odds for finding a particularly favorable mutant. A library may of course also be combined with other libraries or proteinaceous molecules. One such combination may be occasioned by the desire to provide a library comprising an array of mutants of different proteinaceous molecules capable of binding to different epitopes present on a certain target molecule.
An epitope according to the invention is typically present in and/or on a protein produced by a cell. An epitope is a binding site capable of binding said proteinaceous molecule. An epitope may be (part of) any kind of molecule. Typically, an epitope comprises a peptide, a polypeptide, a protein and/or a modification produced by a cellular protein modification machinery.
The cells to which at least part of the library is provided can be living cells and/or dead cells. Typically cells are obtained from a culture. Cells may be processed prior to providing at least part of the library. For instance, for fixation purposes and/or permeabilisation purposes. A functional equivalent of cells is a crude cellular extract. In such an extract the structure of the cells is usually distorted in such a way that individual cells can essentially not be recognised through microscopic means. A crude extract may have undergone several steps to remove one or more undesired components. However, extracts comprising essentially only a proteinaceous molecule comprising said epitope are not considered crude extracts. The division line between what can be considered to be a crude extract and what must be considered to be a purified extract is difficult to give. However, extracts comprising more or less intact organelles are functionally equivalent to cells. A functional equivalent of a cell must comprise most of the epitope in a form essentially similar to a form the epitope has when it is present in and/or on an intact cell comprising said epitope.
Removal of the part of the library that is not bound to the cells and/or the functional equivalent thereof, can be achieved through washing the cells and/or functional equivalent thereof with a suitable solution such as a buffered isotonic solution. Cells can be washed easily by pelleting the cells and suspending the cells in a suitable solution. For removal of that part of the library that is not bound to a functional equivalent of cells, such as an extract of cells, it is advantageous to attach the functional equivalent thereof to a carrier thus enabling easy manipulation of the functional equivalent. Cells may of course also be attached to a carrier. A preferred method of removing unbound proteinaceous molecules is by means of one or more washing steps. It is advantageous to provide for one or more stringent washing steps to remove proteinaceous molecules that are bound with an eventually undesired low affinity. For cells or parts thereof such as organelles and/or membranous particles attachment to a carrier is not required, though may still be advantageous. A method of the invention usually comprises more than 10,000 cells or functional equivalent thereof. However, lower amounts of cells or equivalent thereof may also be used. The invention can even be performed using only one cell.
A proteinaceous molecule may be any proteinaceous molecule capable of binding to an epitope. Non-limiting examples of such a proteinaceous molecule are an antibody (artificial or natural), a FAB-fragment (artificial or natural), a single chain Fv fragment, a T-cell receptor, a ligand, a receptor, a peptide selected preferably from a library for specific epitope binding capacity or a matrix attachment protein. Of course, functional equivalents of said proteinaceous molecules may also be used. Such a functional equivalent comprises the same epitope binding activity in kind not necessarily in amount. A functional equivalent may be a part, a derivative and/or an analogue of said proteinaceous molecule. A derivative is typically obtained through amino acid substitution. A proteinaceous molecule is said to be able to bind to an epitope when cells comprising said epitope, upon exposure to said proteinaceous molecule followed by one or more washing steps, are found to retain said proteinaceous molecule to a significantly higher extend than other cells, essentially not comprising said epitope.
In a preferred embodiment of the invention said proteinaceous molecule comprises a single chain Fv fragment (scFv) and/or a FAB fragment, or a functional equivalent thereof. A functional equivalent of said scFv and/or said FAB fragment is a part, derivative and/or analogue of said scFv and/or said FAB comprising essentially the same binding activity as said scFv and/or FAB fragment in kind not necessarily in amount.
In a preferred embodiment each of said mutants of a proteinaceous molecule is physically linked to a vehicle comprising nucleic acid encoding said mutant proteinaceous molecule. This has the advantage that when said member is recovered from said cells and/or functional equivalent thereof, one simultaneously recovers nucleic acid encoding said proteinaceous molecule. Said nucleic acid is then available for multiplication, analysis, subcloning and/or expression in a cell.
Preferably, said vehicle comprises a virus-like particle such as a phage capsid or a functional equivalent thereof. A virus-like particle is preferred since it is able to condense nucleic acid into a manageable form. A virus-like particle is also preferred for the reason that it may be used to efficiently introduce the nucleic acid of the selected member into a cell. This is particularly advantageous when the nucleic acid, once introduced in the cell, is capable of multiplication, thus allowing for instance the easy isolation of relatively large amounts of said nucleic acid.
In another preferred aspect of the invention said epitope comprises a tumour-associated epitope. A tumour-associated epitope is an epitope essentially characteristic for tumour cells in a body. Said epitope can be present in other cells as long as it is not present in said other cells in the same way as in tumour cells. For instance, an epitope is a tumour-associated epitope when it is present on the surface of a tumour cell and essentially not present on the surface of non-tumour cells due to, for instance but not limited to, a substantially lower expression of said epitope in non-tumour cells. Said epitope may also be present on other cells as long as said cells do not normally co-exist with tumour cells in the same body. A typical example is a tumour-associated epitope present on foetal cells but essentially not present on normal adult cells. A tumour-associated epitope may be individually determined, i.e. a tumour-associated epitope for one individual may not be a tumour-associated epitope in another individual of the same species. A tumour-associated epitope may also be a part of a protein that is present on normal cells but wherein the glycosylation of the protein in normal cells is different from the glycosylation of the protein on tumour cells.
In another aspect the invention provides a molecule capable of binding to said epitope, comprising at least part of a member obtained with a method according to the invention. In one embodiment said part comprises a part of the epitope binding site of said member, or a functional equivalent thereof. In another embodiment said part is a part not directly involved in epitope binding. One example of such a part not directly involved in epitope binding is a part involved in the association with complement factors. Another example is a part associated with tissue penetration of said proteinaceous molecule. This may be due to altered epitope binding properties or due to other mutations. A part can of course comprise more than one property. For instance a part may comprise the epitope binding site and a part involved in association with complement factors. Preferably said molecule comprises an antibody or a functional part thereof. Said antibody is preferably synthesised artificially. Preferably, in a cell cultured in vitro. In one embodiment said antibody is human, humanised and/or human-like, or a functional equivalent thereof.
In another aspect the invention provides the use of a cell and/or a functional equivalent thereof displaying an epitope for obtaining an evolved epitope binding molecule with an enhanced property as compared to the epitope binding molecule said evolved epitope binding molecule is at least in part derived from. In one embodiment said epitope binding molecule comprises a part of a complementarity determining region of an antibody or a functional equivalent thereof. In another embodiment said property comprises an enhanced epitope binding property. In yet another embodiment said property comprises an enhanced tissue penetration property and/or an enhanced complement activation property.
In one embodiment of the invention higher affinity huMabs that bind to the tumor-associated antigen Ep-CAM were obtained by constructing small phage display libraries of mutant scFv antibody fragments derived from the parental anti-Ep-Cam scFv UBS-54. These libraries were subsequently panned on intact Ep-Cam-positive tumor cells. Stringent washing steps during phage selections resulted in the isolation of scFv C52, which was converted into an intact IgGl huMab with a 15-fold affinity improvement and a KD=4*10−10 M. The affinity improvement resulted mainly from a lower koff (or dissociation constant). Light chain shuffling and DNA shuffling were employed to introduce mutations in the antibody V regions. The approximately four fold increase in affinity achieved with each of these mutagenesis approaches were comparable to results achieved by antibody affinity maturation using other mutagenesis and phage display selection techniques (11, 14, 33). In the present invention it is demonstrated that affinity selection can be performed on intact fixed cells, precluding the need to purify or express the target antigen as a recombinant molecule.
Previous selection procedures for isolation of higher affinity antibody variants from phage display libraries have used purified soluble antigen or antigen immobilized on a solid phase as targets for phage selections. It has been noted that selection on solid-phase-bound antigen results in the preferential selection of dimeric over monomeric scFv, due to avidity, thus interfering with the selection of truly higher affinity scFv (14). Selection in solution reduces the avidity effect but requires the careful and step-wise reduction of target antigen concentration in subsequent selection rounds (14). As has been noted for other scFv, in solution scFv UES-54 is a mixture of dimers (30%) and monomers (70%). This ratio was maintained in mutants A37, B43, and C52, showing that the selection on intact cells did not result in a biased selection for dimers (data not shown) Screening for higher affinity binders out of a selected phage pool has been carried out by ranking of mutant scFv according to a lower koff as determined by surface plasmon resonance (6). Although it was attempted to rank the selected scFv according to this method, it was found the experimental data with crude periplasmic scFv preparations was difficult to evaluate due to complexity of the affinity plots (RU versus time) resulting from the mixtures of monomers, dimers and aggregates. Therefore, it was decided to pursue clones that were dominating the phage pools after three rounds of selection. Of note, dominance of phage clones in selections is not entirely determined by affinity but also influenced by scFv expression level, folding efficiency and level of toxicity to E. Coli. Although in the analysis, the presence of dominant clones correlated with higher affinity scFv, it cannot be excluded that other, higher affinity yet less dominant clones were present in the selected phage pools.
Light chain shuffling resulted in the replacement of the original Vk2 light chain by a Vk3 light chain in mutant A37. Structural analysis revealed that the canonical structure of the Vk3 light chain in A37 consisted of a much shorter loop, creating a broader interaction surface. Thermodynamically, it is attractive for antigen-antibody interactions to have a large and close interaction surface because more water molecules are excluded (gain in solvent entropy) and, more importantly, many simultaneous interactions (hydrogen bonds, van der Waals and dipole-dipole interactions) can occur between epitope and paratope (contributing to binding enthalpy) (23). Structurally, this results in a broad face binding site of the antibody that is complementary to an equally broad epitope on a large interacting protein. In contrast, binding sites consisting of deep clefts that snugly surround antigen are generally associated with small ligands such as peptides and low molecular weight organic molecules (23).
DNA shuffling of the VL region but not the VH region resulted in the isolation of higher affinity antibodies. The modeled structure of high affinity huMab C52 indicated that only one of the seven mutations within the VL region, AsnL30A→Ser, most likely directly affects the interaction with Ep-CAM. The other mutations can result in more subtle optimization of the antibody binding site through additional hydrogen bonds and improved packing interactions, as previously reported (21), rather than a specific improved interaction between one of the mutated antibody residues and the antigen Ep-Cam. Indeed, mutation SerL31 results in an additional hydrogen bond with ValL29 that stabilizes the LCDR1 loop. The major gain in affinity appears to be caused by mutations located at the periphery of the antigen combining site in CDR1, resembling the distribution of mutations found in in vivo somatically mutated V regions (34).
Antibody-mediated killing of solid tumors in vivo is a complex process involving Fc receptor-bearing effector cells of the immune system, complement factors and signaling events resulting from the interaction between the antibody and its target. The relative importance and contribution to this process by antibody-related characteristics such as affinity and isotype are becoming the focus of antibody research, spurred by the recent successes of engineered antibodies in the clinic. We exploited the engineered high and lower affinity anti-Ep-CAM huMabs to study two aspects of antibody affinity-dependent processes: killing of tumor cells and penetration of antibodies into clusters of tumor cells mimicking micrometastasis.
It was then found that the lower affinity UBS-54 penetrated faster into the center of the multicell spheroid, resulting in a homogeneous distribution of the huMab. This observation supports other studies showing that very high affinities of antibodies (>109 M−1) lead to trapping of antibody at the tumor edge and slow their penetration into the tumor interior (31, 32, 35, 36).
To our surprise, the lower affinity huMab UBS-54 mediated a persistently higher specific tumor cell lysis with PBMC as effector source compared to the higher affinity mutant C52. The same results were obtained with target cells transfected with an Ep-CAM cDNA construct lacking a cytoplasmic tail, suggesting that signaling via Ep-CAM did not play a role in tumor cell killing. Although many FcγR are able to trigger ADCC, the high affinity FcγRI appears to be the most effective trigger molecule (37,38). We propose that quantitative differences in activation of effector cells mediated via binding of antibodies to high affinity FcγRI may affect their killing capacity in ADCC. Although the mechanism has not been elucidated for FcγRI, recent experiments with the FcεR, another high affinity member the multichain immune recognition receptor family, have shown that aggregation of this receptor by an excess of low-affinity ligand leads to the sequestration of the receptor associated kinase Lyn (39). As a consequence, a smaller number of aggregates simultaneously induced by a higher affinity ligand become deprived of Lyn and are thus unable to initiate the signaling cascade (38). In this model, scarcity of a receptor associated kinase prevents low affinity interactions to activate the complete signaling cascade (40, 41). Based on our in vitro tumor cell killing data we hypothesize that extensive FcγRI receptor triggering by very high affinity antibodies may also result in sequestration of receptor associated kinases and consequently result in a less-efficient FcγRI-mediated induction of the cascade of events leading to activation of effector cells.
The CDCC experiments showed a significantly higher specific tumor cell lysis with huMab C52 compared to huMab UBS-54, indicating an advantage of higher affinity antibodies in activating the complement system. Although the improved capacity of the higher affinity mutant in activating the complement system is evident in vitro, several studies indicate that CDCC may play a marginal role in in vivo tumor cell killing. Most tumor cells express complement-inhibiting regulators which protect the cells against lysis by autologous complement (42-46). Furthermore, tumor cell-specific monoclonal antibodies have been found to be equally effective in eradicating tumors in mice deficient in complement factor C5 as in control mice (47). Thus, ADCC may be the dominant immunological mechanism to kill tumor cells, suggesting that the lower affinity UBS-54 with its higher killing capacity in ADCC may be favorable for passive immunotherapy.